Dm505

The establishment of transgenic mice expressing AKAR3EV (PKAchu mice) was described previously (Kamioka et al., 2012 (link)). Briefly, 8- to 13-week-old female mice were used for the in vivo imaging. The ear hair was removed with a razor 1 d before the experiments. Mice were anesthetized with 1.5% isoflurane (FUJIFILM Wako Pure Chemical Corp.) inhalation and placed in a side-lying position on an electric heater maintained at 37°C. The ear skin was placed on the cover glass. Two-photon excitation microscopy was performed with an FV1200MPE-IX83 inverted microscope (Olympus) equipped with a ×30/1.05 silicon oil-immersion objective lens (XLPLN 25XWMP; Olympus), an InSight DeepSee Ultrafast laser (Spectra Physics), an IR-cut filter (BA685RIF-3), two dichroic mirrors DM505 (Olympus), and two emission filters (BA460-500 for CFP and BA520-560 for YFP) (Olympus). The excitation wavelength was 840 nm.
The animal protocols were approved by the Animal Care and Use Committee of Kyoto University Graduate School of Medicine (approval no. 22063).

See Figs. 1h,i and 2b, Table 1 and Supplementary Table 1b. The following excitation filters, dichroic mirrors and emission filters combined with ND filters were used:
Exciter: 488.0 IF 10 (488 ± 5 nm) (Cheshire Optical)
Dichroic mirror: DM505 (Olympus)
The excitation light density above the objective was 5.6 W cm⁻².
Emitter: BA510IF (510 nm < ) (Olympus) combined with NDX006 (6% transmittance) (Asahi Spectra)

For two-photon excitation microscopy (2PM), we used an FV1200MPE-IX83 inverted microscope (Olympus) equipped with a 30×/1.05 NA silicon oil-immersion objective lens (UPLSAPO 30XS; Olympus), an LCV110-MPE incubator microscope (Olympus) equipped with a 25×/1.05 water-immersion objective lens (XLPLN 25XWMP2; Olympus), and an InSight DeepSee Laser (Spectra Physics). The laser power was set to 3–18%. The scan speed was set between 4–12.5 μs per pixel. Z-stack images were acquired at 1–10 μm intervals. In time-lapse analyses, images were recorded every 1–3 min. The excitation wavelength for CFP was 840 nm. We used an IR-cut filter (BA685RIF-3), two dichroic mirrors (DM505 and DM570), and two emission filters (BA460-500 for CFP and BA520-560 for YFP) (Olympus). Confocal images were acquired with an FV1000/IX83 confocal microscope (Olympus) equipped with a 30×/1.05 NA silicon oil-immersion objective lens (UPLSAPO 30XS; Olympus).

Live-cell epi-fluorescence imaging (Figures 1A, 1B, 2D, and S1–S5) was performed on an IX70 inverted microscope (Olympus, Tokyo, Japan) equipped with an objective lens (60×, UplanApo N.A. 1.40; Olympus). The temperature of the culture medium was maintained at 37°C by controlling with a stage and a microscope objective lens heater with a controller (MI-IBC; Olympus). A cooled CCD camera (ORCA-ER; Hamamatsu Photonics, Hamamatsu, Japan) was used for the acquisition of cell images. The fluorescence images of EGFP and Alexa488 were taken using a sapphire laser (488-nm Model 488-30 CDRH; Coherent), a dichroic mirror (DM505; Olympus), and an emission filter (BA515-550; Chroma Technology, Yokohama Japan). The fluorescence images of Cy3 were taken with a green solid-state laser (532 nm Compass 315M-100; Coherent, Santa Clara, CA), a dichroic mirror (Q565LP; Olympus), and an emission filter (HQ610/75M; Chroma Technology). The fluorescence images of Cy5 and RFP were taken using a red He–Ne laser (633-nm GLS5360; Showa Optronics), a dichroic mirror (660LP; Olympus), and an emission filter (700/75M; Chroma Technology). Fluorescence images were quantitatively analyzed using AQUA-Lite ver. 10 (Hamamatsu Photonics) at various intervals after microinjection.

See Fig. 2c and Supplementary Table 1c. The following excitation filters, dichroic mirrors and emission filters combined with ND filters were used:
Exciter: 488.0 IF 10 (488 ± 5 nm) (Cheshire Optical)
Dichroic mirror: DM505 (Olympus)
The excitation light density above the objective was 5.4 W cm⁻².
Emitter: BA510IF (510 nm < ) (Olympus) combined with NDX006 (6% transmittance) (Asahi Spectra)

The evaluation of PGC colonization and direct Venus fluorescence was analyzed in gonads of 10-day old chicken embryos. For fluorescence microscopy, an Olympus (SZX16) fluorescence stereo microscope equipped with a high-resolution digital camera (Olympus DP74 CMOS), a light source X-Cite 120 Q, and an excitation filter of 460–490 nm, a band pass emission filter of 515–550 nm and a dichroic mirror DM505 (Olympus) were used for detection of Venus fluorescent cells.
The membrane integrity and Venus-fluorescence of sperm (germline chimera) were identified with flow cytometry (Gallios Cytometer 1.2, Beckman Coulter). Ejaculates were collected by dorso-abdominal massage once a week throughout a period of 5 weeks. The ejaculates were directly diluted (1:5–1:10) with HS1 extender to a concentration of 50 × 10⁶ sperm/ml^{53 (link)}. A staining reaction Master mix with 480 µl HS1 extender and 3 µl To-PRO-3 was prepared for each sample. Per reaction 10–20 µl (5 × 10⁵–1 × 10⁶) of diluted sperm were pipetted into the mixture and incubated for 15 min at 17 °C. In total, 100.000 sperm of each ejaculate were evaluated in double. The average (arithmetic mean) of the five measurements per rooster were calculated (Table 3). As a negative control sperm of a non-treated (Venus-negative) rooster was used.

For two-photon excitation microscopy (2PM), we used an FV1200MPE-IX83 inverted microscope (Olympus, Tokyo, Japan) equipped with a 30x/1.05 NA silicon oil-immersion objective lens (UPLSAPO 30XS; Olympus) and an FV1200MPE-BX61WI upright microscope equipped with a 25x/1.05 water-immersion objective lens (XLPLN 25XWMP; Olympus) and an InSight DeepSee Laser (Spectra Physics, Santa Clara, CA, USA). The laser power was set to 8–10% and 2–4% for the observation of the intestine and the skin, respectively [33 (link), 38 (link)]. The scan speed was set at 20 μs/pixel. We used 840-nm light to excite CFP. We used an IR-cut filter (BA685RIF-3), two dichroic mirrors (DM505 and DM570), and two emission filters (BA460-500 for CFP and BA520-560 for YFP) (Olympus). Acquired images were analyzed with MetaMorph software (Universal Imaging, West Chester, PA, USA) as described previously [34 (link), 39 (link)].
Confocal images were acquired with an FV1000/IX83 confocal microscope (Olympus) equipped with a 30x/1.05 NA silicon oil-immersion objective lens (UPLSAPO 30XS; Olympus).